RNA polymerase III (RNAPIII) transcribes short, highly structured non-coding RNAs involved in diverse cellular processes, including translation, transcription regulation, and splicing. The biomedical relevance of RNAPIII activity in humans is wide-ranging: tRNA, 5S rRNA, vault RNA, 7SL RNA, and small NF90-associated RNA (snaR) have been shown to be either elevated or depleted in tumors, neurodegenerative brain tissues, viral- infected B cells, parasite-infected macrophages, and HER2-positive breast cancer cells, respectively. While these and other examples highlight aberrant activities in distinct health-related contexts, a growing body of evidence suggests that multiple RNAPIII-transcribed gene subclasses play important roles in the context of heart development and disease: tRNA and other small RNA levels are altered during early stages of cardiac differentiation and depleted in response to hypoxia, loss of 7SK RNA is sufficient to induce cardiac hypertrophy, RMRP RNA is elevated in mouse models of cardiac hypertrophy and in patients with ischemic heart-failure, and RNAPIII-transcribed Y RNA confers cardioprotection to oxidatively stressed cardiomyocytes. Despite these findings, little is currently known about the functional role of dynamic non-coding RNA levels within these contexts, or whether differences in RNA levels are driven by RNAPIII transcription due in part to several unique challenges related to the sequencing and alignment of short, highly structured and repetitive RNAs. The proposed study seeks to address these deficiencies by developing new high-throughput genomic methods for profiling RNAPIII transcription and applying these strategies in the context of stem cell-to-cardiomyocyte differentiation. In addition, functional experiments will test the role of dynamic RNAPIII patterns within these contexts, and a genetic-background correction method will be developed to enable future comparisons of RNAPIII transcription across diverse cellular contexts, accounting for differences in copy number variation (CNV) in samples of non-identical genomic origin. The proposed study is the next logical step in my progression to becoming an independently funded investigator with an active and successful research program identifying the role and underlying regulatory mechanisms of RNAPIII transcription within diverse cellular contexts and human disease. This project will provide critical training in human stem cell and cardiac biology, CNV detection methodology, and computational biology and statistics, critical skills necessary to establish my research program and accomplish my long-term goals as an independent group leader. Together, results of this study promise to establish improved genomic tools of significant interest to the scientific community, and to yield important insight on the role and regulation of RNAPIII-transcribed genes during cellular differentiation.